Antagonism of propofol anesthesia by alkyl-fluorobenzene derivatives

Despite their frequent use across many clinical settings, general anesthetics are medications with lethal side effects and no reversal agents. A fluorinated analogue of propofol has previously been shown to antagonize propofol anesthesia in tadpoles and zebrafish, but little further investigation of this class of molecules as anesthetic antagonists has been conducted. A 13-member library of alkyl-fluorobenzene derivatives was tested in an established behavioral model of anesthesia in zebrafish at 5 days post fertilization. These compounds were examined for their ability to antagonize propofol and two volatile anesthetics, as well as their interaction with the anesthetic-binding model protein apoferritin. Two compounds provided significant antagonism of propofol, and when combined, were synergistic, suggesting more than one antagonist sensitive target site. These compounds did not antagonize the volatile anesthetics, indicating some selectivity amongst general anesthetics. For the compounds with the most antagonistic potency, similarities in structure and binding to apoferritin may be suggestive of competitive antagonism; however, this was not supported by a Schild analysis. This is consistent with multiple targets contributing to general anesthesia, but whether these are physiologic antagonists or are antagonists at only some subset of the many anesthetic potential targets remains unclear, and will require additional investigation.

Binding of Propofol and Compound 9 to Apoferritin/HSAF 7 Figure S5 Fluorescence of 1-AMA in the presence of Compound 9 7 Figure S6 Elicited Movement Propofol E50 Curves at various concentrations of Compounds 9 and 10 8 Figure S7 1 H NMR (400 MHz) of compound 2 (1-methyl-2-fluorobenzene) 9 Figure S8 GC/MS of compound 2 (1-methyl-2-fluorobenzene) Figure S9 1 H NMR (400 MHz) of compound 4 (1,4-dimethyl-2-fluorobenzene) Figure S10  Behavioral Experiments with Volatile Anesthetics.Solutions of inhaled volatile anesthetics were stored in glass vials with minimal head space and were transferred utilizing Hamilton gas-tight syringes to prevent evaporative loss.Isoflurane (Primal Care) was stored at 4°C, and sevoflurane (AbbVie Inc.) was stored at -20°C.Solutions were made in DMSO and then diluted in glass scintillation vials to the desired experimental concentrations in E3 (embryo water).Each vial contained 22 mL of E3 which minimized head space to prevent drug loss.Vials were sealed quickly after addition of volatile anesthetics to minimize loss.Solutions were mixed via vortexing, and anesthetics dissolved readily in the final DMSO concentration of 2%.For behavior experiments the zebrafish were placed in the 96 well glass plate (Zissner North America 3600500), and E3 solutions containing drug were quickly pipetted into each well, with slight over filling to prevent entrapment of air bubbles during application of adhesive strips (3M 300LSE).The zebrafish were allowed to equilibrate in the solution for 10 minutes prior to observation for 22 minutes in the behavior system.Glass 96-well plates were inverted (adhesive side down) when placed in the behavior system.As shown in Fig. S25, the presence of the adhesive did not have a significant change in zebrafish behavior.To assure consistent concentration of volatile anesthetic, this experiment was repeated without fish and concentrations were measured by HPLC.See methods below and Fig. S26.

High Performance Liquid Chromatography (HPLC) of Volatile Anesthetics in Glass 96-well
Plates.The methodology used for behavior experiments was repeated to prepare the substances for high pressure liquid chromatography.The drug was then removed from the wells with a Hamilton Gas-Tight syringe (without taking the adhesive strip off) and dispensed into a glass HPLC vial, that were tightly sealed and wrapped in parafilm.The concentration of volatile anesthetic was measure in multiple wells at time points from 15-180 minutes as compared to a standard curve of each anesthetic.Analysis of HPLC occurred immediately after each time point (15-minute increments).Standard solutions of volatile anesthetic in methanol were freshly prepared prior to each experiment.Samples were injected into the Beckman Coulter HPLC system equipped with an analytical Eclipse Plus C18 column (Agilent Technologies) and Waters 2410 refractive index detector.The mobile phase was a single solution of acetonitrile and 20 mM phosphate buffer (pH 4) at 23:77 v/v at a flow rate of 1.0 mL/min.Chromatic peaks for the substances were identified by retention time and compared to standard curves.
1-Aminoanthracene Competition Fluorescence Assay.This assay was performed in a similar manner to that previously described with a few modifications. 4,5A saturated solution of 1-AMA (1aminoanthracene, Sigma-Aldrich) was prepared by sonication in a 1x phosphate buffered saline (PBS, pH = 7.4) followed by filtration with a 0.22 μm polyvinylidene fluoride filter (PVDF, Rephile).After filtration, the 1-AMA concentration was determined by UV−vis spectroscopy utilizing an experimentally determined extinction coefficient of 1-AMA (Σ368 = 4073 M −1 cm −1 ). 4 Stock solutions of ligand were prepared in DMSO.Horse spleen apoferritin (HSAF, Sigma-Aldrich, 0.2 μm filtered) was used as received.1-AMA (15 μM assay concentration) was preequilibrated with HSAF (15 μM assay concentration of dimer), and 5 μL of ligand stock in DMSO was then added for a final volume of 500 μL (1% DMSO v/v in PBS).Ligand concentrations ranged from 1 to 125 μM except where solubility limited the upper range.Upon addition of the ligand, the samples were mixed and immediately analyzed with a spectrofluorometer (Shimadzu RF-5301 PC) with an excitation wavelength of 380 nm and emission detection from 400 to 700 nm.The fluorescence curves were corrected by subtracting contributions from 1-AMA alone and HSAF alone.Fluorescence intensities at 515 nm were plotted as a percentage of the control (1-AMA and HSAF bound with no competing ligand present) and were fitted to a logarithmic four parameter variable slope using GraphPad Prism (version 9.4.0,GraphPad Software, La Jolla California, USA).The result of these experiments can be seen in Figure 4 in the main text.From the screening of PFAL Compounds (data presented in Fig. 2 from the main text), estimated EC 50 values for spontaneous movement (SM) were calculated (with upper and lower constraints at 100 and 0 respectively).More formal EC50 values were determined for the the most potent compounds (9 and 10).This data can be seen in the main text (Fig. 6).To test for fluorescent interference by a library compound via inner filter effect or other mechanism, a control study was done with compound and 1-AMA alone (no apoferritin/HSAF is present in these samples).At concentrations higher than those used in the 1-AMA assay for compound 9 (see Figure 4 in the main text), some enhancement of fluorescence was found, but this would not account for the differences in the binding/displacement curves seen with PFAL members.Peak area: 36@2.6min,97@3.1min,81@3.6min,27@4.3min,5975@4.5minPurity based on Peak area (total two injections): 96.12% and 95.81%In order to create a sealed environment for testing zebrafish behavior with volatile anesthetics, glass 96-well plates were sealed with 3M 300LSE adhesive and inverted prior to observation in the Daniovision chamber.In order to confirm that there were no significant behavioural changes due to the presence of this seal, a comparison was made between this method and a standard open well-plate used in experiments with non-volatile compounds.Spontaneous movement over 10 minutes was recorded for zebrafish in 2% DMSO either in an open glass 96-well plate, or in the same plate sealed with the 3M 300LSE adhesive.No statistical difference was noted between the groups when compared with a Welch's t-test.To test the effectiveness of the 3M 300LSE adhesive to prevent loss of the sevoflurane and isoflurane, the concentration was monitored over time by HPLC.The recovery of these anesthetics over 3 hours is shown in the graphs above.Sevoflurane concentration remained constant during the 3-hour testing period, but there was a slow loss of isoflurane over time with 96% of the original concentration remaining at the 3-hour mark.This finding may be due in part to their relative vapor pressures (sevoflurane: 157 mmHg, isoflurane: 238 mmHg at 20°C).Behavioural experiments using this setup occur with a 30-minute incubation period, and thus this method of sealing the plates with an adhesive strip is sufficient for this purpose with both anaesthetics.

Fluorobenzene ( 1 ) 1 -Figure S1 :Figure S2 .
Figure S1: Toxicity of PFAL Compounds Alone and with Propofol.A) Toxicity of Propofol only and each PFAL compound alone (no propofol), with each PFAL compound showing much less toxicity than Propofol.Mean and standard deviation are shown for each data point.B) Comparison of LD 50 values from Panel A. LD 50 and 95% CI are shown for each data point.Due to limitations in PFAL solubility and the resultant limitations in data collection, 95% CI was unable to be determined for 1 and 11 and no upper limit of 95% CI was determined for 8 and 10.C) To further characterize the toxicity of the PFAL compounds, they were co-administered at 30 μM compound with variable concentrations propofol.This showed a small increase in toxicity compared to administration of propofol alone.D) Comparison of LD 50 values from panel C. Comparison of LD 50 values from Panel C. 95% CI are shown for each data point.E) Calculated LD 50 s from Panel A and C. ns: not significant, P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, ****: P ≤ 0.0001

Figure S3 .
Figure S3.Estimated Propofol EC 50 Values.From the screening of PFAL Compounds (data presented in Fig.2from the main text), estimated EC 50 values for spontaneous movement (SM) were calculated (with upper and lower constraints at 100 and 0 respectively).More formal EC50 values were determined for the the most potent compounds (9 and 10).This data can be seen in the main text (Fig.6).

Figure S5 .
Figure S5.Fluorescence of 1-AMA in the presence of Compound 9.To test for fluorescent interference by a library compound via inner filter effect or other mechanism, a control study was done with compound and 1-AMA alone (no apoferritin/HSAF is present in these samples).At concentrations higher than those used in the 1-AMA assay for compound 9 (see Figure4in the main text), some enhancement of fluorescence was found, but this would not account for the differences in the binding/displacement curves seen with PFAL members.

Figure S4 .
Figure S4.Binding of Propofol and Compound 9 to Apoferritin/HSAF.In the same 1-AMA/HSAF assay seen in Figure 4 in the main text, this graph shows the similarity in the binding curves of propofol and compound 9.

Figure S25 .
Figure S25.Effect of 3M Adhesive on 5 dpf Zebrafish Movement.In order to create a sealed environment for testing zebrafish behavior with volatile anesthetics, glass 96-well plates were sealed with 3M 300LSE adhesive and inverted prior to observation in the Daniovision chamber.In order to confirm that there were no significant behavioural changes due to the presence of this seal, a comparison was made between this method and a standard open well-plate used in experiments with non-volatile compounds.Spontaneous movement over 10 minutes was recorded for zebrafish in 2% DMSO either in an open glass 96-well plate, or in the same plate sealed with the 3M 300LSE adhesive.No statistical difference was noted between the groups when compared with a Welch's t-test.

Figure S26 .
Figure S26.Concentration of Sevoflurane and Isoflurane in the Glass 96-well Plates Over Time.To test the effectiveness of the 3M 300LSE adhesive to prevent loss of the sevoflurane and isoflurane, the concentration was monitored over time by HPLC.The recovery of these anesthetics over 3 hours is shown in the graphs above.Sevoflurane concentration remained constant during the 3-hour testing period, but there was a slow loss of isoflurane over time with 96% of the original concentration remaining at the 3-hour mark.This finding may be due in part to their relative vapor pressures (sevoflurane: 157 mmHg, isoflurane: 238 mmHg at 20°C).Behavioural experiments using this setup occur with a 30-minute incubation period, and thus this method of sealing the plates with an adhesive strip is sufficient for this purpose with both anaesthetics.